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Interpretive Summary: To protect consumer health, food producers and government regulators require rapid, sensitive, and accurate methods to detect harmful bacteria (pathogens) in food. One of the most popular methods for the detection of foodborne pathogens, the polymerase chain reaction (or PCR), is based on the amplification of specific DNA sequences associated with the pathogen of interest. Recently, microscopic magnetic particles (nanoparticles) have been used to capture and concentrate DNA prior to PCR, allowing for increased detection sensitivity. Unfortunately, conflicting studies have revealed that nanoparticles have either a positive or negative effect on PCR. The current study was undertaken to carefully examine the effect of nanoparticles on PCR. The results revealed mechanisms by which nanoparticles affect PCR and the consequences for PCR-based pathogen detection. These results illuminate some limitations associated with nanoparticle-based DNA capture and PCR, and will allow the development of more sensitive and accurate PCR-based assays for the detection of foodborne pathogens.

Technical Abstract: Nanomaterials have been widely reported to affect the polymerase chain reaction (PCR). However, many studies in which these effects were observed were not comprehensive, and many of the proposed mechanisms have been primarily speculative. In this work, we used amino-modified silica-coated magnetic nanoparticles (ASMNPs, which can be collected very easily using an external magnetic field) as a model and compared them with gold nanoparticles (AuNPs, which have been studied extensively) to reveal the mechanisms by which nanoparticles affect PCR. We found that nanoparticles affect PCR primarily by binding to PCR components causing inhibition and affecting PCR specificity, efficiency and yield. We found that: (1) Excess nanomaterials inhibit PCR by adsorbing to DNA polymerase, Mg2+, oligonucleotide primers, or DNA templates. Nanoparticle surface-active groups are particularly important to this effect. (2) a) Nanomaterials do not inhibit nonspecific amplification products caused by false priming as previously surmised. It was shown that relatively low concentrations of nanoparticles inhibited the amplification of long amplicons, and increasing the amount of nanoparticles inhibited the amplification of short amplicons. This concentration phenomenon appears to be the result of the formation of “joints” upon the adsorption of ASMNPs to DNA templates. b) Nanomaterials are able to inhibit nonspecific amplification products due to incomplete amplification by preferably adsorbing single-stranded incomplete amplification products. (3) Some types of nanomaterials, such as AuNPs, enhance the efficiency and yield of PCR because these types of nanoparticles can adsorb to single- stranded DNA more strongly than to double-stranded DNA. This behavior assists in the rapid and thorough denaturation of double-stranded DNA templates. Therefore, the interaction between the surface of nanoparticles and PCR components is sufficient to explain most of the effects of nanoparticles on PCR.